Trans-ethnic study confirmed
independent associations of
HLA-A
*
02:06
and
HLA-B
*
44:03
with cold
medicine-related Stevens-Johnson
syndrome with severe ocular surface
complications
Mayumi Ueta
1,2, Chitra Kannabiran
3, Tais Hitomi Wakamatsu
4, Mee Kum Kim
5, Kyung-Chul Yoon
6,
Kyoung Yul Seo
7, Choun-Ki Joo
8, Virender Sangwan
9, Varsha Rathi
9, Sayan Basu
9, Almas Shamaila
3,
Hyo Seok Lee
6, Sangchul Yoon
7, Chie Sotozono
1, Jose´ A´lvaro Pereira Gomes
4, Katsushi Tokunaga
10& Shigeru Kinoshita
11Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan,2Research Center for Inflammation and
Regenerative Medicine, Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan,3Prof Brien Holden Eye Research Centre, L V Prasad Eye Institute, Hyderabad, India,4Department of Ophthalmology, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil,5Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea,6Department of
Ophthalmology, Chonnam National University, Gwangju, Korea,7Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea,8Department of Ophthalmology & Visual Science, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea,9Cornea and Anterior Segment Services, L V Prasad Eye Institute, Hyderabad, India,10Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), are acute
inflammatory vesiculobullous reactions of the skin and mucous membranes. Cold medicines including
non-steroidal anti-inflammatory drugs and multi-ingredient cold medications are reported to be important
inciting drugs. Recently, we reported that cold medicine related SJS/TEN (CM-SJS/TEN) with severe
mucosal involvement including severe ocular surface complications (SOC) is associated with
HLA-A
*
02:06
and
HLA-B
*
44:03 in the Japanese. In this study, to determine whether HLA-B
*
44:03 is a common risk
factor for CM-SJS/TEN with SOC in different ethnic groups we used samples from Indian, Brazilian, and
Korean patients with CM-SJS/TEN with SOC, and investigated the association between CM-SJS/TEN with
SOC and
HLA-B
*
44:03 and/or HLA-A
*
02:06. We found that HLA-B
*
44:03 was significantly associated
with CM-SJS/TEN with SOC in the Indian and Brazilian but not the Korean population, and that
HLA-A
*
02:06 might be weakly associated in the Korean- but not the Indian and Brazilian population.
S
tevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN) with spots, are
acute inflammatory vesiculobullous reactions of the skin and mucous membranes such as the ocular
surface, oral cavity, and genitals. They are rare but often associated with inciting drugs and/or infectious
agents
1–3.
The association between human leukocyte antigen (HLA) genotypes and drug-induced severe cutaneous
adverse reactions (SCARs) including SJS/TEN has been reported. There was a strong association between
HLA-B
*
58:01
and SCARs, including SJS/TEN and the drug-induced hypersensitivity syndrome (DIHS),
induced by the uric acid lowering drug allopurinol. This association was observed in Han Chinese-
4,
Caucasian-
5, and Japanese patients
6, suggesting that different ethnic groups share the same risk factor(s)
for allopurinol-induced SCARs.
HLA-B
*
15:02
exhibited a very strong association with
carbamazepine-OPEN
SUBJECT AREAS:
GENETICS RESEARCH
RISK FACTORS
Received
2 June 2014
Accepted
15 July 2014
Published
7 August 2014
Correspondence and requests for materials should be addressed to M.U. (mueta@koto. kpu-m.ac.jp)
induced SJS/TEN in Taiwanese Han Chinese patients
7and
HLA-A
*
31:01
was strongly associated with carbamazepine-induced
SCARs including SJS/TEN in Japanese-
8and European patients
9.
We recently reported that cold medicine-related SJS/TEN with
severe mucosal involvement including severe ocular surface
com-plications (SOC) is associated with
HLA-A
*
02:06
and
HLA-B
*
44:03
in Japanese patients
10.
The ophthalmologists Mondino et al.
11and the dermatologists
Roujeau et al.
12,13reported that
HLA-B12
(HLA-Bw44) was
signifi-cantly increased in Caucasian SJS patients many of whom developed
SJS/TEN after taking non-steroidal anti-inflammatory drugs
(NSAIDs).
HLA-B12
is primarily coded by
HLA-B
*
44:02
or
HLA-B
*
44:03
(http://www.allelefrequencies.net/). The significant
asso-ciation between HLA-B12 and SJS/TEN in Caucasian patients may
be attributable to their genetic background.
To determine whether
HLA-B
*
44:03
is a common risk factor for
CM-SJS/TEN with SOC in different ethnic groups we used samples
from Indian, Brazilian, and Korean patients with CM-SJS/TEN with
SOC, and investigated the association between CM-SJS/TEN with
SOC and
HLA-B
*
44:03
and/or
HLA-A
*
02:06.
Methods
Our study was approved by the institutional review boards of the participating institutions. All experimental procedures were conducted in accordance with the principles of the Helsinki Declaration. The purpose of the research and the experi-mental protocols were explained to all participants, and their prior written informed consent was obtained.
Patients and controls.Ophthalmologists diagnosed SJS/TEN based on a confirmed
history of acute-onset high fever, serious mucocutaneous illness with skin eruptions, and involvement of at least two mucosal sites including the ocular surface1,14,15. They
defined patients with SOC as those who manifested a pseudomembrane and an epithelial defect on the ocular surface in the acute stage, and as patients with ocular sequelae such as dry eye, trichiasis, symblepharon, and conjunctival invasion into the cornea in the chronic stage.
As in our previous study, we focused on SJS/TEN with SOC suspected of having been induced by cold medicines such as multi-ingredient cold medications and NSAIDs. As we found earlier that the genetic predisposition might be different between SJS/TEN with and without severe mucosal involvement including SOC10we
focused on patients from different ethnic groups who presented with SJS/TEN with SOC.
Samples from Indian patients with CM-SJS/TEN were collected at the LV Prasad Eye Institute (n520; 12 males, 8 females; age range 7 to 63 years; median age 27.16
13.4 (SD) years). Their age at onset ranged from 3 to 42 years (median age at onset, 19.2612.2 (SD) years; in 8 patients the age at onset was unknown). The drugs administered to these patients and the HLA type (A and B) of patients with CM-SJS/ TEN with SOC are shown in Supplemental Table 1. The specific drug(s) were not known in all patients. Healthy volunteers (n555; 29 males, 26 females; median age 36.0611.6 years) served as the Indian controls.
Samples from Brazilian patients with CM-SJS/TEN were collected at the Federal University of Sao Paulo (n539, 15 males, 24 females; age range 13 to 69 years; median age, 37.1615.9 years; age range at onset, 3 to 69 years; median age at onset, 24.06
17.2 years). The drugs administered, the ethnicity, and the HLA type (A and B) of these CM-SJS/TEN patients with SOC are shown in Supplemental Table 2. Healthy volunteers (n5134; 55 males, 79 females; median age 41.2612.8 years) were the Brazilian controls (ethnicity: pardo, n566; white, n562; black, n54, Indian plus white, n52).
Samples from Korean patients with CM-SJS/TEN were collected at the Seoul National University College of Medicine, Chonnam National University, Yonsei University, and the Catholic University of Korea. There were 31 patients (12 males, 19 females) ranging in age from 4 to 71 years (median age 33.7619.0 years). Their age at SJS/TEN onset ranged from 3 to 63 years (median age at onset, 23.0616.1 years). The drugs used and the HLA type (A and B) of these patients with SOC are presented in Supplemental Table 3. The specific drug(s) were not known in all patients. Healthy volunteers (n590; 35 males, 55 females; median age 31.767.9 years) were the Korean controls.
Samples from Indian subjects were obtained by extracting DNA from whole peripheral blood with the phenol chloroform method. For Brazilian samples, DNA was extracted from whole peripheral blood using the PAX gene blood DNA kit (Qiagen, Hilden, Germany) or from saliva using Oragene DNA (Kyodou International, Kanagawa, Japan). To obtain the samples from Korean subjects, DNA was extracted from whole peripheral blood using the PAXgene Blood DNA kit (Qiagen).
HLA genotyping.For the analysis ofHLA-AandHLA-Bwe performed polymerase
chain reaction (PCR) assays followed by hybridization with sequence-specific oligonucleotide probes using commercial bead-based typing kits (Wakunaga,
Table
1
|Results
of
association
analyses
in
patients
with
CM-SJS/TEN
with
SOC
ethnic group HLA genotyp e Car rier freq uency (%) Dominant model anal ysis Gene frequ ency (%) Domin ant mod el analysis CM-SJS/T EN with SOC Control P Pc Odds ratio CM-SJS/TEN with SOC Contr ol P Pc Odds ratio (95% CI) (95 % CI) Indian A * 02:06 1/20 (5.0% ) 3/55 (5.5% ) 0.938 -0.9 1 (0.09 –9.31) 1/39 (2.5%) 3/11 0 (2.7% ) 0.9 39 -0.9 1 (0.09 –9.06) B * 44 :03 12/2 0 (60 .0%) 6/55 (10.9%) 1.07 .E-05 2.14. E-05 12.25 (3.57 – 42. 01) 17/40 (42.5%) 7/11 0 (6.4% ) 9. 37.E-08 1. 87.E-07 10.88 (4.04 – 29. 3) Brazilian A * 02:06 0/39 (0.00 %) 0/134 (0.00%) -0/78 (0.00%) 0/26 8 (0.00 %) -B * 44 :03 10/3 9 (25 .6%) 15/134 (11.2%) 0.0239 0 .0478 2.74 (1.12 – 6.7 1) 11/78 (14.1%) 15/2 68 (5.60 %) 0.012 1 0.0242 2.77 (1.22 – 6. 31) Kore an A * 02:06 11/3 1 (35 .5%) 14/90 (15.6%) 0.0181 0 .0362 3.00 (1.18 – 7.5 7) 12/62 (19.4%) 16/1 80 (8.9% ) 0.026 3 0.052 6 2.46 (1.09 – 5. 54) B * 44 :03 6/31 (19 .4%) 18/90 (20.0%) 0.938 -0.9 6 (0.34 –2.69) 7/62 (11.3%) 19/1 80 (10.6 %) 0.8 72 -1.0 7 (0.43 –2.70) P: P values obtained with the x 2-test (Pearson), C I: Confid ence interval. Pc: P values corrected for the multiplic ity of testing by the number of comparisons 2 (HLA-A * 02:06 1 HLA-B * 44:03). CM-SJS/T EN: cold medicine-related S JS/T EN, SOC: severe ocular surfac e complications.www.nature.com/
scientificreports
Hiroshima, Japan). Briefly, the target DNA was PCR-amplified with biotinylated primers specifically designed for amplified exons 2 and 3 of HLA-A, and -B genes. Then the PCR amplicon was denatured and hybridized to complementary oligonucleotide probes (72 probes for HLA-A, 93 probes for HLA-B) immobilized on fluorescent-coded microsphere beads. At the same time, the biotinylated PCR product was labeled with phycoerythrin-conjugated streptavidin and immediately examined with Luminex 100 (Luminex, Austin, TX, USA). Genotype determination and data analysis were performed automatically using the WAKFLOW typing software (Wakunaga, Hiroshima, Japan) according to the manufacturer’s instructions.
Statistical analysis.We compared the carrier frequency and gene frequency of
individual HLA alleles in the patients and controls with thex2-test (Pearson) (JMP
version 11 software; SAS Institute Japan Ltd., Tokyo, Japan).
Results
Strong association between
HLA-B
*
44:03
and CM-SJS/TEN with
SOC in Indian patients
.
We genotyped HLA-A and HLA-B in
samples from Indian subjects (20 CM-SJS/TEN with SOC patients
and 55 controls). Although the number of Indian subjects was small,
we found a strong and significant association between their CM-SJS/
TEN with SOC and
HLA-B
*
44:03
(carrier frequency: p
5
1.07
3
10
25, odds ratio (OR)
5
12.25, gene frequency: p
5
9.37
3
10
28, OR
5
10.88) but not
HLA-A
*
0206
(Table 1).
Significant association between
HLA-B
*
44:03
and CM-SJS/TEN
with SOC in Brazilian patients
.
Next we genotyped HLA-A and
HLA-B in samples from Brazilian subjects (39 CM-SJS/TEN with
SOC patients and 134 controls). Although the number of Brazilian
subjects was small we found a significant association between
Brazilian patients with CM-SJS/TEN with SOC and
HLA-B
*
44:03
(carrier frequency: p
5
0.0239, OR
5
2.74, gene frequency: p
5
0.0121, OR
5
2.77) but not
HLA-A
*
0206
which is absent in the
Brazilian population (Table 1). Interestingly, in Caucasians in the
Brazilian samples (Brazilian Caucasian CM-SJS/TEN with SOC
patients: n
5
15, Brazilian Caucasian controls: n
5
62), the
association with
HLA-B
*
44:03
was stronger (carrier frequency:
p
5
0.0037, OR
5
6.22, gene frequency: p
5
0.0011, OR
5
5.99).
Association between
HLA-A
*
02:06
and Korean patients with
CM-SJS/TEN with SOC
.
We also genotyped HLA-A and HLA-B in
samples from Koreans (31 patients with CM-SJS/TEN with SOC
and 90 controls). Although the number of Korean patients was
small we found a significant association between patients with
CM-SJS/TEN with SOC and
HLA-A
*
0206
(carrier frequency: p
5
0.0362, OR
5
3.00, gene frequency: p
5
0.0263, OR
5
2.46) but not
HLA-B
*
44:03
(Table 1).
Discussion
We previously reported that in the Japanese, CM-SJS/TEN with
severe mucosal involvement including SOC was associated with
HLA-A
*
02:06
and
HLA-B
*
44:03
10. In the present study we
investi-gated whether the association with these alleles is shared by other
ethnic groups. We found that
HLA-B
*
44:03
was strongly associated
with CM-SJS/TEN with SOC in the Indian population which is
gen-etically close to European populations
16and significantly associated
in the Brazilian population which is comprised of individuals with
different ethnic backgrounds. There was no association between
HLA-B
*
44:03
and CM-SJS/TEN with SOC in the Korean population.
HLA-A
*
02:06
was weakly associated in the Korean population which
is genetically close to the Japanese, but not in the Indian and Brazilian
population.
HLA-B12
(HLA-Bw44) was significantly increased in Caucasian
SJS patients many of whom developed SJS/TEN after taking
NS-AIDs
11–13. Because
HLA-B12
is primarily coded by
HLA-B
*
44:02
or
HLA-B
*
44:03
(http://www.allelefrequencies.net/), the significant
association of
HLA-B12
with SJS/TEN in Caucasian patients may
be attributable to the association with the
HLA-B
*
44:03
genotype.
We also found that in Brazilian Caucasian patients with CM-SJS/
TEN with SOC, the significant association with
HLA-B
*
44:03
was
stronger than in the entire study population of Brazilians with
CM-SJS/TEN with SOC. To determine whether
HLA-B
*
44:03
is a
com-mon marker for CM-SJS/TEN with SOC in Caucasian, HLA analysis
of European patients with CM-SJS/TEN with SOC is needed.
Although
HLA-A
*
02:06
was strongly associated with the Japanese
CM-SJS/TEN with SOC, and the Korean and Japanese population is
genetically close
16, in Korean patients CM-SJS/TEN with SOC was
not strongly associated with
HLA-A
*
02:06. To determine whether
HLA-A
*
02:06
is a common marker for CM-SJS/TEN with SOC in
East Asian populations further investigations using a larger number
of samples are needed.
We also performed a meta-analysis by adding our
previously-reported samples
10. We used Cochran-Mantel-Haenszel statistics
and found that both HLA-A
*
02:06 and HLA-B
*
44:03 are
signifi-cantly associated with CM-SJS/TEN with SOC (Supplemental
Table 4).
SCARs including SJS/TEN and DIHS induced by allopurinol were
commonly and strongly associated with
HLA-B
*
58:01
in patients of
different ethnic backgrounds including Han Chinese-
4, Caucasian-
5,
and Japanese patients
6. This observation suggests that different
eth-nic groups share the same risk factor(s) for allopurinol-induced
SCARs.
With respect to carbamazepine-induced SJS/TEN, different HLA
alleles are associated.
HLA-B
*
15:02
is associated in Taiwanese Han
Chinese patients
7and
HLA-A
*
31:01
in Japanese-
8and European
patients
9.
In CM-SJS/TEN with SOC, the associated alleles we identified are
HLA-A
*
02:06
in Japanese and Korean patients and
HLA-B
*
44:03
in
Indian-, Brazilian-, and Japanese patients. Studies are underway to
determine whether other HLA alleles are associated with CM-SJS/
TEN with SOC in other populations.
1. Ueta, M.et al. Toll-like receptor 3 gene polymorphisms in Japanese patients with Stevens-Johnson syndrome.Br J Ophthalmol91, 962–965 (2007).
2. Yamane, Y., Aihara, M. & Ikezawa, Z. Analysis of Stevens-Johnson syndrome and toxic epidermal necrolysis in Japan from 2000 to 2006.Allergol Int56, 419–425 (2007).
3. Yetiv, J. Z., Bianchine, J. R. & Owen, J. A. Jr. Etiologic factors of the Stevens-Johnson syndrome.South Med J73, 599–602 (1980).
4. Hung, S. I.et al. HLA-B*5801 allele as a genetic marker for severe cutaneous adverse reactions caused by allopurinol.Proc Natl Acad Sci U S A102, 4134–4139 (2005).
5. Lonjou, C.et al. A European study of HLA-B in Stevens-Johnson syndrome and toxic epidermal necrolysis related to five high-risk drugs.Pharmacogenet Genomics18, 99–107 (2008).
6. Tohkin, M.et al. A whole-genome association study of major determinants for allopurinol-related Stevens-Johnson syndrome and toxic epidermal necrolysis in Japanese patients.Pharmacogenomics J13, 60–69 (2013).
7. Chung, W. H.et al. Medical genetics: A marker for Stevens-Johnson syndrome.
Nature428, 486 (2004).
8. Ozeki, T.et al. Genome-wide association study identifies HLA-A*3101 allele as a genetic risk factor for carbamazepine-induced cutaneous adverse drug reactions in Japanese population.Hum Molec Genetics20, 1034–1041 (2011).
9. McCormack, M.et al. HLA-A*3101 and carbamazepine-induced hypersensitivity reactions in Europeans.N Engl J Med364, 1134–1143 (2011).
10. Ueta, M.et al. Independent strong association of HLA-A*02:06 and HLA-B*44:03 with cold medicine-related Stevens-Johnson syndrome with severe mucosal involvement.Sci Rep4, 4862 (2014).
11. Mondino, B. J., Brown, S. I. & Biglan, A. W. HLA antigens in Stevens-Johnson syndrome with ocular involvement.Arch Ophthalmol100, 1453–1454 (1982). 12. Roujeau, J. C.et al. HLA phenotypes and bullous cutaneous reactions to drugs.
Tissue Antigens28, 251–254 (1986).
13. Roujeau, J. C.et al. Genetic susceptibility to toxic epidermal necrolysis.Arch Dermatol123, 1171–1173 (1987).
14. Ueta, M.et al. Association between prostaglandin E receptor 3 polymorphisms and Stevens-Johnson syndrome identified by means of a genome-wide association study.J Allergy Clin Immunol126, 1218–1225 e1210 (2010).
15. Ueta, M., Sotozono, C., Tokunaga, K., Yabe, T. & Kinoshita, S. Strong association between HLA-A*0206 and Stevens-Johnson Syndrome in the Japanese.Am J Ophthalmol143, 367–368 (2007).
www.nature.com/
scientificreports
16. Abdulla, M. A.et al. Mapping human genetic diversity in Asia.Science326, 1541–1545 (2009).
Acknowledgments
This work was conducted as part of the BioBank Japan Project supported by the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government, and as part of the Promotion Project of Knowledge-Based Industrial Clustering of Okinawa Prefecture. It was supported in part by grants-in-aid for scientific research from the Japanese Ministry of Health, Labour and Welfare, a research grant from the Kyoto Foundation for the Promotion of Medical Science, and the Intramural Research Fund of Kyoto Prefectural University of Medicine. The funding agencies had no role in the study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.
Author contributions
M.U. wrote the main manuscript text and prepared the tables. M.U., C.K., T.W., M.K., K.Y., K.S., C.J., V.S., V.R., S.B., A.S., H.L., S.Y., C.S., J.G., K.T. and S.K. contributed to material of the research and reviewed the manuscript.
Additional information
Supplementary informationaccompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
How to cite this article:Ueta, M.et al. Trans-ethnic study confirmed independent
associations ofHLA-A*02:06andHLA-B*44:03with cold medicine-related Stevens-Johnson syndrome with severe ocular surface complications.Sci. Rep.4, 5981; DOI:10.1038/srep05981 (2014).
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http:// creativecommons.org/licenses/by-nc-nd/4.0/
